Amplex® Red reagent technology

Amplex® Red reagent is a colorless substrate that reacts with hydrogen peroxide (H2O2) with a 1:1 stoichiometry to produce highly fluorescent resorufin (excitation/emission maxima=570/585 nm) (Figure 1). Amplex® Red reaction can be used to routinely detect as little as 10 picomoles of H2O2 in a 100 µL volume (50 nM, Figure 2), at least a 10-fold greater sensitivity than that attained with the commonly used scopoletin assay for hydrogen peroxide. In the scopoletin assay, HRP catalyzes conversion of the fluorescent scopoletin to a nonfluorescent product. Unlike scopoletin, the Amplex® Red reagent is a fluorogenic substrate with very low background fluorescence. Consequently, assays using Amplex® Red as the substrate result in an increase in fluorescence, not a decrease—an inherently superior method for enzymatic assays. Amplex® UltraRed reagent—the second-generation Amplex® substrate—exhibits increased resistance to oxidation and works better in lower pH environments. It is used in assays that have an acid intermediate resulting in a lower reaction pH (i.e., myeloperoxidase using the EnzChek® MPO Activity Assay Kit).

Other advantages of the Amplex® Red reaction over scopoletin-based H2O2 assays include high chemical stability of the Amplex® Red reagent and its fluorescent product, resorufin, and the long-wavelength spectra of resorufin. Because resorufin has excitation/emission maxima of ~570/585 nm (versus 360/460 nm for scopoletin), there is much less interference from autofluorescence in most biological samples.

Figure 1. Principle of coupled enzymatic assays using our Amplex® Red reagent. Oxidation of glucose by glucose oxidase results in generation of H2O2, which is coupled to conversion of the Amplex® Red reagent to fluorescent resorufin by HRP.